Dental products comprising bone growth enhancing peptide

ABSTRACT

Dental products such as toothpastes, mouthwash and dental floss are disclosed which products are enhanced by having dissolved, dispersed or coated thereon a compound which promotes bone growth. Preferred compounds are peptide sequences comprising 10 to 50 amino acids are disclosed. The sequences are characterized by containing an integrin binding motif such as RGD sequence and the remainder of amino acids contiguous with the RGD sequence in matrix extracellular phosphoglycoprotein. The sequences may be formulated for dispersed in toothpaste or a mouthwash and administered to enhance bone/tooth growth. When the dental products are used repeatedly over time they enhance good dental health.

TECHNICAL FIELD

The invention relates generally to the field of dental products and moreparticularly to such products supplemented so that they are useful intreating skeletal diseases.

BACKGROUND OF THE INVENTION

A wide range of dental products including toothpastes, mouthwashes anddental floss are used. These products are generally intended to reducedental diseases. However, it is well-documented that disorders ofskeletal tissues and mineral metabolism cause numerous significanthealth problems and such can be specific to dental problems.

In humans, the maximum bone mass occurs between the age of 15 and 40 andis referred to as “peak bone mass.” After such peak bone mass age, bonemass begins declining gradually and the mechanical strength of the boneis accordingly reduced. Consequently, when mechanical strength declinesto a certain level, the individual is at greater risk of bone fracture.This natural occurrence is called osteoporosis if severe enough to bepathogenic.

The speed at which bone loss occurs differs among individuals, andespecially with respect to gender. In females, the speed of bone lossaccelerates immediately after menopause (See FIG. 1) because of asignificant decline in available estrogen, a hormone which plays acritical role in maintaining healthy bone metabolism. Postmenopausalosteoporosis constitutes an important clinical problem because itafflicts significant numbers of women. Notably, the ratio of female tomale osteoporosis patients is 3:1.

The majority of bone diseases are characterized by loss of boneminerals, weakening of bones and consequently, an increase of thefrequency and severity of bone fractures, which are called “pathologicalfracture.” In the elderly population, this has significant socialramifications as well, as many of those with bone fractures havedifficulty with mobility, which often leads to the deterioration ofother mental and physical functions, resulting in dementia, muscularweakness and/or fatigue. In addition, morbidity and pain aresignificantly increased by thrombotic events, such as pulmonary embolismwhich occur as a result of hip or pelvic fractures.

In the United States alone, it is said that 52 million women over age of45 will suffer from osteoporosis by 2000. Current worldwide osteoporosispopulation is around 200 million. Annual incidence of pathologicalfracture in the United States alone is approximately 1.5 million. It isestimated that annual medical costs for those osteoporosis patients inthe United States and world are $14 billion and $60 billion,respectively.

Renal failure is also a significant health problem related to mineralmetabolism and skeletal formation, and the number of its patients isincreasing rapidly. Renal function is declining gradually over severalto ten years period in these patients. When the renal function becomesapproximately a quarter (¼) of the healthy level, the patients areclassified to chronic renal failure. When it becomes approximately onesixth (⅙) thereof, they need to start dialysis and are called end stagerenal disease (ESRD). In patients with chronic renal failure, serumlevels of important minerals such as calcium and phosphate lose theirnormal homeostasis, which results in malformation of skeleton. It iscalled renal osteodystropny (ROD), which is a secondary osteoporosisfrom renal failure. ROD can also cause pathological fracture likeosteoporosis. The prevalence of end stage renal disease (ESRD) in theUnited States is rapidly increasing and about to reach 300 thousand in2000. As ESRD is a part of chronic renal failure, there should be muchhigher number of ROD patients.

There are several other diseases of skeletal tissues and mineralmetabolism such as Paget's Disease, rikets, osteopetrosis,hyperparathyroidism, and so forth and number of patients are affected bythese diseases.

Metabolically, bone is a highly active organ with bone resorption andformation occurring continuously (remodeling). Bone resorption isfacilitated by osteoclasts which are differentiated frommonocyte/macrophage lineage cells. Osteoclasts adhere to the surface ofbone and degrade bone tissue by secreting acids and enzymes. Osteoblastsfacilitate bone formation by adhering to degraded bone tissue andsecreting bone matrix proteins, which are mineralized mostly by calciumand phosphate. Osteoblasts differentiate into bone cells (osteocytes),and become a part of bone tissue.

Numerous experimental approaches have been attempted to eitheraccelerate bone formation or diminish bone resorption. For example,growth factors such as BMPs (bone morphogenetic proteins), TGFβ(transforming growth factor β), IGF (insulin-like growth factor),fibroblast growth factor (FGF) are known to have potent biologicalactivities in bone formation. In particular, a few subfamily moleculesof BMP such as BMP-2 are regarded one of the most potent growth factorsfor hard tissue. However, these factors have not been developed astherapeutic agents for systemic bone diseases. It is because none ofthem can be delivered to the bone selectively and some of these factorssuch as BMPs convert soft tissue into hard tissue. It is called ectopiccalcification and a critical adverse effect for them when they are usedsystemically. Further, the processes of bone formation and resorptionare so closely connected and that makes selective increase of boneformation or selective inhibition of bone resorption extremelydifficult.

Currently, there is a need for an effective treatment for bone loss.Therapeutic agents such as estrogen, calcitonin, vitamin D, fluoride,Iprifravon, bisphosphonates, and a few others have failed to provide asatisfactory means of treatment. (Gennari et al., Drug Saf. (1994)11(3):179-95).

Estrogen and its analogues are frequently administered to patients withpostmenopausal osteoporosis. Estrogen replacement therapy involvesadministration of estrogen just prior to or after the onset ofmenopause. However, as is often the case with steroid hormones, the longterm use of estrogen has significant adverse effects such as breast andother gynecological cancers (Schneider et al., Int. J. Fertil.Menopausal Study (1995) 40(1):40-53).

Calcitonin, an endogenous hormone produced by the thyroid, bindsselectively to osteoclasts, via its receptor, and inactivates them.Since the osteoclast is the only cell which can dissolve bone tissue,calcitonin binding can block or slow down bone degradation caused by theosteoclast. However, this biological mechanism is very short-lived, asthe osteoclasts become tolerant to this drug relatively quickly.Therefore, the use of calcitonin does not provide an effectivetherapeutic option.

Fluoride has been shown to increase bone mass when it is administered tohumans. However, while bone mass is increased, mechanical strength isnot. Therefore, despite the increase in apparent bone mass, the risk offracture remains (Fratzl et al., J. Bone Mineral Res. (1994)9(10):1541-1549). In addition, fluoride administration has significanthealth risks.

Iprifravon has been used to treat osteoporosis in limited areas in theworld. However, the actual efficacy of this compound is questionable andit is not widely accepted as a useful therapeutic agent for bonediseases.

Bisphosphonates are compounds derivatized from pyrophosphate. Synthesisinvolves replacing an oxygen atom situated between two phosphorus atomswith carbon and modifying the carbon with various substituents. Whilebisphosphonates are known to suppress bone resorption, they have littleeffect on bone formation. Furthermore, bisphosphonates adhere to thebone surface and remain there for very long time causing a long-termdecrease in bone tissue turnover. As bone tissue needs to be turned overcontinuously, this decrease in turnover ultimately results in bonedeterioration (Lufkin et al., Osteoporos. Int. (1994) 4(6):320-322;Chapparel et al., J. Bone Miner. Res. (1995) 10(1):112-118).

Another significant problem with the agents described above is that withthe exception of fluoride and iprifravon, they are unsuitable for oraladministration, and thus, must be given parenterally. Since bonedisorders are often chronic and require long-term therapy, it isimportant that therapeutic agents be suitable for oral administration.

In summary, a significant need exists for a therapeutic agent which canprevent or treat bone loss. In particular, a new drug that canselectively increase bone formation and/or number of osteoblast withoutaffecting bone resorption or soft tissue is highly desired.

Another major health problem relating to skeleton and mineral metabolismis that with teeth. In the United States alone, it is estimated that 67million people are affected by periodontal disease and that the annualcost of its treatment is approximately $6.0 billion in 2000. It is said90% of the entire population experience dental caries in their lives.The annual cost to treat them is over $50 billion per year in the UnitedStates alone.

Dental caries is a universal disease and affects children and adults.Periodontal disease, on the other hand, affects mostly adults, and inparticular, the aged. In many cases, the patient's gum is inflamed anddestroyed, the alveolar bone that supports the teeth is deteriorated.Cement that composes the core of the root is also damaged, andsubsequently, teeth fall out. One of the most common treatments fortooth loss involves the use of a dental implant. An artificial implant(osseointegrated dental implants) is placed in the space where the toothwas lost. In severe cases, an entire denture is replaced by implants.However, implants frequently loosen, or fall out because their fixationon the alveolar bone is not always successful. Since alveolar bone issomehow damaged in these patients, the implant can not always besupported well by alveolar bone. When alveolar bone is severely damaged,autogenous bone grafting is made. In this case, a bone graft taken fromanother skeletal tissue of the same patient is grafted in the damagedalveolar area so that the hard tissue is regenerated and sinus iselevated there. Since these treatments require expensive bio-compatiblematerials and/or highly skilled techniques, the cost of treatment isusually very high.

It is believed that dental caries is caused by acidic condition in theoral cavity. For instance, sugars are converted to acid and dissolve thesurface of the teeth. Although only enamel and a part of dentin isaffected in many cases, the damage can reach the pulp cavity in severecases that cause significant pain. The most typical treatment is fillingthe caries lesion with undegradable materials such as metals or metaloxide. Treatment of dental caries mostly depends upon those materialsand the techniques by the dentists, which is often expensive.

Although a few therapeutic agents have been developed and used in dentalarea, they are generally only anti-inflammatory drugs, analgesics, andantibiotics. No generally effective therapeutic agent that directlyimproves periodontal hard tissues has been developed. Obviously, thereis a significant demand for a therapeutic agent that promotesregeneration of alveolar bone and/or teeth, and increases the number andactivity of odontoblasts/osteoblasts that help form of dental tissues.

SUMMARY OF THE INVENTION

Dental products including toothpaste, mouthwash, and dental floss aredisclosed which products are comprised of a compound which enhance bonegrowth. The compound is any of a class of compounds which are useful intreating or preventing a condition associated with skeletal loss orweakness. The compounds are peptides or analogs thereof which comprisebetween 10 and 50 monomer (e.g. amino acids) units. The amino acidsequence comprises an integrin binding motif sequence which may be inthe D- or L-conformation. The remaining monomer units (the sequenceother than the integrin binding motif) in the compound may be amino acidanalogs but are preferably naturally occurring amino acids having asequence which is substantially the same as an amino acid sequencecontiguous with the RGD sequence in the naturally occurring protein,matrix extracellular phosphoglycoprotein (Rowe et. al., Genomics (2000)67:56-68).

An aspect of the invention is a set of peptides and/or peptide analogs.

Another aspect of the invention is to provide toothpaste which comprisesa sufficient concentration of a compound of the invention to enhancetooth and/or alveolar bone growth on areas where deterioration hasoccurred.

Yet another aspect of the invention is to provide a mouthwash whichcomprises a sufficient concentration of a compound of the invention toenhance tooth and/or alveolar bone growth on areas where deteriorationhas occurred.

Still another aspect of the invention is a dental floss having coatedthereon and/or embedded therein a compound of the invention in an amountsuch that repeated application to teeth and/or alveolar bone results inenhanced tooth and/or alveolar bone growth on areas where deteriorationhas occurred.

A feature of the invention is that a compound of the invention comprisedan binding motif sequence in a D or L conformation.

An advantage of the invention is that a compound of the inventionenhances skeletal growth.

Another advantage of the invention is that a compound of the inventionenhances the amount of osteoblast and possibly odontoblast cells on thesurface of new skeletal growth.

Another aspect of the invention is to provide a formulation fortherapeutic use which comprises a sufficient concentration of a compoundof the invention and can be injected into the pulp of teeth, the spacebetween the root of teeth and gum, or alveolar bone to prevent thedamage on teeth and/or alveolar bone or regenerate the hard tissue inthe damaged teeth and/or alveolar bone.

An object of the invention is to provide a method of treating skeletalloss by the administration/application of any formulation/composition ofthe invention.

These and other objects, aspects, features and advantages will becomeapparent to those skilled in the art upon reading this disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the relationship between bone mass and age inhumans.

FIG. 2 is a schematic drawing of a matrix extracellularphosphoglycoprotein wherein the area designated as “A” includessequences which match peptides of the present invention and the areadesignated as “B” is a highly homologous motif to a group of bone-toothmatrix phosphoglycoproteins such as osteopontin (OPN), dentinsialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and bonesialoprotein II (IBSP).

FIGS. 3A, 3B, 3C, and 3D are actual photographs of bone cross-sections(from a seven day mouse calvaria organ culture study) showing theeffects of a control FIG. 3A), fibroblast growth factor-I (FGF-1) FIG.3B), and two peptides of the invention designated D-00004 and D-00006(FIGS. 3C and 3D, respectively).

FIG. 4 is a graph comparing the effects of different compounds oncalvaria.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the toothpastes, mouthwashes, dental floss products, peptides,analogs, formulations, and methodology of the present invention aredescribed, it is to be understood that this invention is not limited toany particular embodiment described, as such may, of course, vary. It isalso to be understood that the terminology used herein is with thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “apeptide” includes a plurality of such peptides and reference to “themethod” includes reference to one or more methods and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

The term “dental product” refers to all and any product used in themouth. Preferably the product is used on a regular basis by consumerssuch as toothpaste, mouthwash and dental floss. However, the termincludes products used solely by oral surgeons and dentists such asdental implants and materials used to fill dental cavities.

The terms “treat”, “treating”, “treatment” and the like are usedinterchangeably herein and mean obtaining a desired pharmacologicaland/or physiological effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of partially or completely curing a diseaseand/or adverse effect attributed the disease such as enhancing theeffect of vitamin D. “Treating” as used herein covers treating a diseasein a vertebrate and particularly a mammal and most particularly a human,and includes:

(a) preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;

(b) inhibiting the disease, i.e. arresting its development; or

(c) relieving the disease, i.e. causing regression of the disease.

The invention is particularly directed towards peptides which make itpossible to treat patient's which have experienced bone loss or whichwould be expected to experience bone loss and thus is particularlydirected towards preventing, inhibiting, or relieving the effects ofbone loss. A subject is “treated” provided the subject experiences atherapeutically detectable and beneficial effect which may be measuredbased on a variety of different criteria including increased bonegrowth, increased bone strength or other characteristics generallyunderstood by those skilled in the art to be desirable with respect tothe treatment of diseases related to bone.

The term “antibody” is meant an immunoglobulin protein capable ofbinding an antigen. The term “antibody” as used herein is intended toinclude antibody fragments (e.g. F(ab′)₂, Fab′, and Fab) capable ofbinding an antigen or antigenic fragment of interest.

The term “binds specifically” is meant high avidity and/or high affinitybinding of an antibody to a specific peptide—specifically a peptide ofthe invention. Antibody binding to its specific target epitope isstronger than the binding of the antibody to other epitopes on thepeptide or to other epitopes on other peptides. Antibodies which bindspecifically to a peptide of interest may be capable of binding to otherpeptides at a weak, yet detectable level (e.g. 10% or less of thebinding shown to the peptide of interest). Such weak binding orbackground binding, is readily discernable from the specific antibodybinding to the peptide of interest, e.g. by the use of appropriatecontrols.

The term “skeletal loss” refers to any situation in which skeletal mass,substance or matrix or any component of the skeleton, such as calciumand phosphate, is decreased or the bone is weakened such as in terms ofits ability to resist being broken.

The term “skeleton” includes both bone and teeth. In the same manner,the term “skeletal” means both bone and teeth.

The term “osteoporosis” is intended to refer to any condition involvingbone loss, i.e. involving a reduction in the amount of bone mass orsubstance resulting from any cause. The term particularly results in abone loss resulting from demineralization of the bone, post menopausalor peri-menopausal estrogen decrease or nerve damage.

The term “subject” refers to any vertebrate, particularly any mammal andmost particularly including human subjects.

Invention in General

In general the invention comprises any dental product comprising acompound which enhances bone growth. The product is preferably atoothpaste, mouthwash or dental floss. The compound is preferably apeptide comprising from 10 to 50 amino acids. The amino acids arepreferably one of the twenty naturally occurring L-amino acids. However,D-amino acids may be present as may amino acid analogs. A sequence ofthe invention will comprise an integrin binding motif such as RGDsequence in either the L- or D-form but preferably in theL-conformation. The peptide of the invention can be amidated ornon-amidated on its C-terminus, or carboxylated or non-carboxylated onits N-terminus. The peptide of the invention may or may not contain aglycosaminoglycan binding motif such as SGDG sequence in L- or D-isomerform. A compound of the invention is still further characterized bybiological activity i.e. it enhances skeletal growth as well as thegrowth or recruiting of osteoblast or odontoblast cells on surface ofthe new skeletal growth.

Specific Dental Products

The present invention is broadly applicable to all types of dentalproducts and is particularly useful in connection with products used byconsumers on a regular basis such as toothpaste, mouthwash and dentalfloss.

Specific examples of toothpastes which could be modified by having acompound of the invention dissolved, suspended or mixed therein includethose toothpaste compositions disclosed and described in U.S. Pat. Nos.6,045,780; 5,951,966; 5,932,193; 5,932,191; and 5,876,701. These patentsas well as the patents and publications cited in these patents areincorporated herein by reference for the purpose of disclosing anddescribing various toothpaste compositions which can be used inconnection with the present invention.

Compounds of the invention can also be used in combination with alltypes of mouthwashes. The various compounds including specific peptidesdisclosed herein can be dissolved or dispersed within a wide range ofdifferent compositions including the mouthwash compositions disclosedand described within U.S. Pat. Nos. 5,993,785; 5,817,295; 5,723,106;5,707,610; 5,549,885; 5,470,561; 5,466,437; 5,455,023; 5,407,664;5,328,682; and 5,256,401 all of which are incorporated herein byreference along with the patents and publications cited therein in orderto disclose and describe various mouthwash compositions useful inconnection with the present invention.

Compounds of the invention can also be coated on or absorbed intovarious types of filament materials used as dental flosses. Specificexamples of dental floss materials which can be used in combination withthe present invention include those disclosed and described within U.S.Pat. Nos. 6,102,050; 6,080,481; 6,027,592; 6,026,829; 6,016,816;5,967,155; 5,937,874; 5,915,392; 5,904,152; 5,875,797; and 5,845,652 allof which are incorporated herein by reference along with the patents andpublications cited therein in order to disclose and describe dentalfloss filament materials which can be used in combination with thepresent invention.

Specific Peptides

Specific examples of peptides of the invention comprise seven toforty-seven amino acids on either side of the RGD sequence of thenaturally occurring sequence of matrix extracellularphosphoglycoprotein. Thus, examples of peptides of the inventioncomprising sequences taken from the following sequence and including theRGD sequence shown in bold:

(SEQ ID NO:1) DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQERGDNDISPFSGDGQPFKDIPGKGEATGPDLEGKDIQTGFAGPSEAESTHL

Specific examples of peptides of the invention which comprise the RGDsequence as the terminal sequence include the following:

(SEQ ID NO:2) AQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQERGD (SEQ IDNO:3) RGDAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:4)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDRGD (SEQ ID NO:5)RGDSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:6)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGRGD (SEQ ID NO:7)RGDTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:8)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFERGD (SEQ ID NO:9)RGDLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:10)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSRGD (SEQ ID NO:11)RGDLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:12) DSQAQKSPVKSKSTHRIQHNIDYLKHLSKRGD(SEQ ID NO:13) RGDVKKIPSDFEGSGYTDLQE (SEQ ID NO:14)DSQAQKSPVKSKSTHRIQHNIDYLKRGD (SEQ ID NO:15) RGDIPSDFEGSGYTDLQE (SEQ IDNO:16) DSQAQKSPVKSKSTHRIQHNIDRGD (SEQ ID NO:17) RGDDFEGSGYTDLQE (SEQ IDNO:18) DSQAQKSPVKSKSTHRRGD (SEQ ID NO:19) RGDGSGYTDLQE (SEQ ID NO:20)DSQAQKSPVKRGD (SEQ ID NO:21) RGDGYTDLQE (SEQ ID NO:22) DSQAQKSRGD (SEQID NO:23) RGDNDISPFSGDGQPFKDIPGKGEATGPDLEGKDIQTGFA

Specific examples of the peptides of the invention which comprise theRGD internally include the following:

(SEQ ID NO:24) NDI RGDSPFSGDGQPFKDIPGKGEATGPDLEGKDIQTGFA (SEQ ID NO:25)NDISPF RGDSGDGQPFKDIPGKGEATGPDLEGKDI (SEQ ID NO:26) NDISPFSGDRGDGQPFKDIPGKGEATGPDL (SEQ ID NO:27)FSGDGQPFKDIPGKGEATGPDLEGKDIQTGFAGPSEAES RGDTHL (SEQ ID NO:28)IPGKGEATGPDLEGKDIQTGFAGPSE RGDAESTHL (SEQ ID NO:29) EATGPDLEGKDIQTGFAGRGDPSEAESTHL (SEQ ID NO:30) NDISPFSGDGQPFKD RGDIPGKGEATGPDLEGK (SEQ IDNO:31) GKGEATGPDLEGKDI RGDQTGFAGPSEAESTHL (SEQ ID NO:32)FSGDGQPFKDIPGKGEATG RGDPDLEGKDIQTGFAGPSEA (SEQ ID NO:33)DGQPFKDIPGKGEATG RGDPDLEGKDIQTGF (SEQ ID NO:34) PFKDIPGKGEATGRGDPDLEGKDIQ (SEQ ID NO:35) DIPGKGEATG RGDPDLEGKDIQTGFAGP (SEQ ID NO:36)DGQPFKDIPGKGEATG RGDPDLEGKDIQTGF (SEQ ID NO:37) GKGEATGRGDPDLEGKDIQTGFAGPSEA (SEQ ID NO:38) EATG RGDPDLEGKDIQTGF (SEQ ID NO:39)EATG RGDPDLEGK (SEQ ID NO:40) EATG RGDPDL

All or any of the amino acids in the above sequences may be in the D- orL-conformation and may be substituted with equivalent analogs. Thepreferred embodiments comprise naturally occurring amino acids in theL-conformation.

All or any of the above sequences may be amidated or non-amidated ontheir C-terminus, or carboxylated or non-carboxylated on theirN-terminus.

Matrix extracellular phosphoglycoprotein was cloned and characterizedfrom a human tumor that caused osteomalacia in the patients. Thisextremely rare type of tumor called Oncogenic HypophosphatemicOsteomalacia (OHO) tumor has been known to cause renal phosphate leak,hypophosphatemia (low serum phosphate levels), low serum calcitriol(1,25-vitamin D3), and abnormalities in skeletal mineralization(Osteomalacia). In the patients of OHO tumor, resection of the tumorsresults in remission of all of the above symptoms and it has beenproposed that a circulating phosphaturic factor secreted from OHO tumorplays a role in osteomalacia. Matrix extracellular phosphoglycoproteinwas proposed as a candidate of this phosphaturic factorphosphoglycoprotein (Rowe et. al., Genomics (2000) 67:56-68).

Phosphate plays a central role in many of the basic processes essentialto the cell and the mineralization of skeleton. In particular, skeletalmineralization is dependent on the regulation of phosphate and calciumin the body and any disturbances in phosphate-calcium homeostasis canhave severe repercussions on the integrity of bone. In the kidney,phosphate is lost passively into the glomerular filtrate and is activelyreabsorbed via a sodium (Na+) dependent phosphate cotransporter. In theintestine, phosphate is absorbed from foods. A sodium (Na+) dependentphosphate cotransporter was found to be expressed in the intestine andrecently cloned (Hilfiker, PNAS 95(24) (1998), 14564-14569). The liver,skin and kidney are involved in the conversion of vitamin D3 to itsactive metabolite, calcitriol, which plays an active role in themaintenance of phosphate balance and skeletal mineralization.

Vitamin D deficiency causes rickets in children and osteomalacia inadults. Both conditions are characterized by failure of calcification ofosteoid, which is the matrix of skeleton.

Thus, all of the humoral functions by matrix extracellularphosphoglycoprotein, namely, renal phosphate leak, hypophosphatemia (lowserum phosphate levels), low serum calcitriol (1,25-vitamin D3), areharmful to healthy skeletal formation.

Matrix extracellular phosphoglycoprotein is a large polypeptide with 525amino acid with short N-terminus signal sequence. Therefore, it ishighly probable that this molecule is secreted from its producing cellsinto the body fluid and circulation. Out of its 525 amino acid sequence,23 amino acid motif on the C-terminus showed high similarities to agroup of bone-tooth mineral matrix phosphoglycoproteins such asosteopontin (OPN), dentin sialophosphoprotein (DSPP), dentin matrixprotein 1 (DMP1), and bone sialoprotein II (IBSP). It has been proposedthat these bone-tooth mineral matrix phosphoproteins may play importantroles in skeletal mineralization.

Notwithstanding the above observations about matrix extracellularphosphoglycoprotein, smaller peptide sequence containing integrinbinding motif that is located within the amino acid sequence and farfrom its C-terminus sequence with a high degree of similarity to otherbone-tooth mineral matrix phosphoglycoproteins demonstrated a verypotent skeletal formation activity and increased the number ofosteoblasts on such skeletal formation surface. The potency of suchactivities was equivalent to fibroblast growth factor (FGF). It wassurprising in that small motifs within a large protein which protein hasdestructive functions on the skeleton demonstrated potent bone formationactivity, and that such motifs were located far from the sequence whichshowed homology to other known bone-tooth matrix proteins.

Another surprising fact was that potent skeletal formation motifs of theinvention contained an integrin binding motif, in particular, RGDsequence. It has been reported that a synthetic peptide containing theRGD sequence inhibited bone formation and resorption in a mineralizingorgan culture system of fetal rat skeleton (Gronowicz et. al. Journal ofBone and Mineral Research 9(2):193-201 (1994)), that is a very similarexperimental method used to test the subject of the present invention.

Further, the skeletal formation activity provided by the small peptidesof the invention was as potent as that of an intact growth factor suchas FGF.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Synthesis of D-00001, Etc.

Six different peptides were manually synthesized by the9-fluorenylmethoxycarbonyl (Fmoc) strategy and prepared in theC-terminal amide form. The six peptides are as follows:

D-00001: IPSDFEGSGYTDLQE (SEQ ID NO:41) D-00002: DFEGSGYTDLQERGD (SEQ IDNO:42) D-00003: YTDLQERGDNDISPF (SEQ ID NO:43) D-00004: ERGDNDISPFSGDGQ(SEQ ID NO:44) D-00005: NDISPFSGDGQPFKD (SEQ ID NO:45) D-00006:TDLQERGDNDISPFSGDGQPFKD (SEQ ID NO:46) (C-terminus amidated)Amino acid derivatives and resins were purchased from Bachem, Inc.,Torrance, Calif., and Novabiochem, La Jolla, Calif. The respective aminoacids were condensed manually in a stepwise manner using4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin. N-methylpyrrolidone was used during the synthesis as a solvent. Forcondensation, diisopropylcarbodiimide/N-hydroxybenzotriazole wasemployed, and for deprotection of N^(α)-Fmoc groups, 20% piperidine inN-methyl pyrrolidone was employed. The following side chain protectinggroups were used: Asn and Gln, trityl; Asp, Glu, Ser, and Thr, t-butyl;Arg, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; and Lys, t-butoxycarbonyl.Resulting protected peptide resins were deprotected and cleaved from theresin using a trifluoroaceticacid-thioanisole-m-cresol-ethanedithiol-H₂O (80:5:5:5:5, v/v) at 20° C.for 2 h. Resulting crude peptides were precipitated and washed withethyl ether then purified by reverse-phase high performance liquidchromatography (using Vydac 5C18 column and a gradient ofwater/acetonitrile containing 0.1% trifluoroacetic acid). All peptideswere obtained with 5-20% yield (from the starting resin). Purity of thepeptides was confined by analytical high performance liquidchromatography. Identity of the peptides was confirmed by a Sciex APIIIIE triple quadrupole ion spray mass spectrometer.

Example 2 Fetal Mouse Calvarial Assay Reagents

FGF-1 was purchased from Peprotech Inc. (Rocky Hill, N.J.). RGD-1, 2, 3,4, 5 and 6 (referred to here as D-00001, D-00002, D-00003, D-00004,D-00005 and D-00006) were provided by Dr. Nomizu (Hokkaido University,Japan).

Mice

Pregnant ICR mice were purchased from SLC Japan Co. Ltd. (Shizuoka,Japan).

Mouse Calvarial Organ Culture

Mouse calvarial organ culture was performed as described in Mundy G etal. Science 286: 1946-1949, 1999 and Traianedes K et al. Endocrinology139: 3178-3184, 1998. The calvaria from 4-days-old mice were excised andcut in half along the sagittal suture. Each half of the calvaria wasplaced on a stainless steel grid in a 12-well tissue culture dish (AsahiGlass Techno Corp., Funabashi, Japan). Each well contained 1.5 ml of BGjmedium (Sigma, St. Louis, Mo.) supplemented with 0.1% bovine serumalbumin (Sigma) and each compound. FGF-1 was used as a positive controlas described by Mundy et al. The medium was changed at day 1 and 4, andthe assay was terminated at day 7.

Histomorphometrical Analysis

Calvaria was fixed with 10% neutral-buffered formalin, decalcified with4.13% EDTA and embedded in paraffin. 4 mm-thickness sections were madeand stained with hematoxylin and eosin. New bone area was measured usingImage-Pro Plus (Media Cybernetics, Silver Spring, Md.).

The six peptides of Example 1 were tested for their ability to enhancebone growth with the tests being carried out as described above inExample 2. The peptides which did not include the RGD sequence did notshow positive results. The other four peptides showed positive resultswith the best results being obtained with the sequences

D-00004: ERGDNDISPFSGDGQ, (SEQ ID NO:44) and D-00006:TDLQERGDNDISPFSGDGQPFKD. (SEQ ID NO:46)The best results are in FIG. 3 (specifically FIGS. 3C and 3D). Data fromthese results are graphically shown in FIG. 4.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-15. (canceled)
 16. A method of treating bone tissue of a patient,comprising: administering to the patient a therapeutically effectiveamount of a formulation comprising: a pharmaceutically acceptablecarrier and a peptide consisting of ten to fifty amino acids, whereinthe peptide is comprised of an RGD integrin binding motif and furtherwherein the peptide is characterized by biological activity whichenhances bone growth; and allowing the formulation to enhance bothgrowth in the patient.
 17. The method of claim 16, wherein the peptideis contiguous with an RGD sequence of naturally occurring protein matrixextracellular phosphoglycoprotein.
 18. The method of claim 16, whereinthe peptide consists of 15 to 35 amino acids.
 19. The method of claim16, wherein the amino acids are in an L-conformation.
 20. The method ofclaim 16, wherein the peptide is chosen from: DFEGSGYTDLQERGD (SEQ IDNO:42) YTDLQERGDNDISPF (SEQ ID NO:43) ERGDNDISPFSGDGQ (SEQ ID NO:44)TDLQERGDNDISPFSGDGQPFKD. (SEQ ID NO:46)


21. A method of claim 16, wherein the peptide is TDLQERGDNDISPFSGDGQPFKD(SEQ ID NO:46).
 22. The method of claim 16, wherein the administering isby injection.
 23. The method as claimed in claim 16, wherein the peptideis selected from the group consisting of: (SEQ ID NO:2)AQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQERGD (SEQ ID NO:3)RGDAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:4)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDRGD (SEQ ID NO:5)RGDSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:6)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGRGD (SEQ ID NO:7)RGDTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:8)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFERGD (SEQ ID NO:9)RGDLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:10)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSRGD (SEQ ID NO:11)RGDLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:12) DSQAQKSPVKSKSTHRIQHNIDYLKHLSKRGD(SEQ ID NO:13) RGDVKKIPSDFEGSGYTDLQE (SEQ ID NO:14)DSAQKSPVKSKSTHRIQHNIDYLKRGD (SEQ ID NO:15) RGDIPSDFEGSGYTDLQE (SEQ IDNO:16) DSQAQKSPVKSKSTHRIQHNIDRGD (SEQ ID NO:17) RGDDFEGSGYTDLQE (SEQ IDNO:18) DSQAQKSPVKSKSTHRRGD (SEQ ID NO:19) RGDGSGYTDLQE (SEQ ID NO:20)DSQAQKSPVKRGD (SEQ ID NO:21) RGDGYTDLQE (SEQ ID NO:22) DSQAQKSRGD (SEQID NO:23) RGDNDISPFSGDGQPFKDIPGKGEATGPDLEGKDIQTGFA (SEQ ID NO:24) NDIRGDSPFSGDGQPFKDIPGKGEATGPDLEGKDIQTGFA (SEQ ID NO:25) NDISPFRGDSGDGQPFKDIPGKGEATGPDLEGKDI (SEQ ID NO:26) NDISPFSGDRGDGQPFKDIPGKGEATGPDL (SEQ ID NO:27)FSGDGQPFKDIPGKGEATGPDLEGKDIQTGFAGPSEAES RGDTHL (SEQ ID NO:28)IPGKGEATGPDLEGKDIQTGFAGPSE RGDAESTHL (SEQ ID NO:29) EATGPDLEGKDIQTGFAGRGDPSEAESTHL (SEQ ID NO:30) NDISPFSGDGQPFKD RGDIPGKGEATGPDLEGK (SEQ IDNO:31) GKGEATGPDLEGKDI RGDQTGFAGPSEAESTHL (SEQ ID NO:32)FSGDGQPFKDIPGKGEATG RGDPDLEGKDIQTGFAGPSEA (SEQ ID NO:33)DGQPFKDIPGKGEATG RGDPDLEGKDIQTGF (SEQ ID NO:34) PFKDIPGKGEATGRGDPDLEGKDIQ (SEQ ID NO:35) DIPGKGEATG RGDPDLEGKDIQTGFAGP (SEQ ID NO:36)DGQPFKDIPGKGEATG RGDPDLEGKDIQTGF (SEQ ID NO:37) GKGEATGRGDPDLEGKDIQTGFAGPSEA (SEQ ID NO:38) EATG RGDPDLEGKDIQTGF (SEQ ID NO:39)EATG RGDPDLEGK (SEQ ID NO:40) EATG RGDPDL.


24. A method of treating bone tissue of a patient, comprising:administering to the patient a therapeutically effective amount of aformulation comprising: a pharmaceutically acceptable carrier and apeptide consisting of 15 to 35 amino acids in an L-conformation, whereinthe peptide is comprised of an RGD integrin binding motif and furtherwherein the peptide is characterized by biological activity whichenhances bone growth; and allowing the formulation to enhance bothgrowth in the patient.
 25. The method of claim 24, wherein the peptideis contiguous with an RGD sequence of naturally occurring protein matrixextracellular phosphoglycoprotein.